The overall goal of this research is to understand how telomere proteins regulate telomerase recruitment and other the steps in telomere replication, but yet ensure that the chromosome terminus is protected from unwanted DNA repair or processing activities during the remainder of the cell cycle. The research is pertinent to human health because loss of telomere capping leads to chromosome fusions and chromosome rearrangements, the underlying cause of many forms of cancer. Defects in telomerase action are also associated with human disease as the resulting telomere shortening can lead to dyskeratosis congenita and pulmonary fibrosis. Telomere protection is generally achieved by multi-protein complexes that bind the telomeric DNA and sequester the DNA terminus. During replication, some of the same proteins regulate processing of the telomeric DNA, recruit telomerase, or stimulate telomerase activity. However, the mechanism by which individual proteins regulate these processes is still poorly understood. We will address this question through aims 1 and 3 which examine how three newly identified Tetrahymena telomere proteins, p61, p45 and Pot1b, regulate access to telomerase, enhance telomerase activity, or promote new telomere synthesis. Aim 2 addresses the dynamic nature of telomere structure. Telomeres must undergo a structural switch during S-phase in order to make the chromosome terminus accessible. Despite the importance of this structural change, the underlying cause is not understood. We will tackle this problem by examining Tetrahymena telomere composition and how this changes during the cell cycle or under different growth conditions. We will use Tetrahymena for our work because it has unique traits that make it exceptionally well suited for the genetic and biochemical approaches needed to address the above questions. In particular, it has ~40,000 telomeres per cell, correspondingly high levels of telomerase, and telomeres that exist as discrete complexes which can be purified from bulk chromatin. The specific aims are as follows. Aim 1: Determine the function of p61 and p45 in telomere capping and telomerase regulation. Aim 2: Characterize Tetrahymena telomere composition and dynamics. Aim 3: Determine the role of Pot1b during new telomere synthesis. The outcome should be a comprehensive picture of the role played by individual telomere proteins in telomere protection, replication, and chromosome healing, and of how the dynamic changes in telomere organization promote these processes. PUBLIC HEALTH RELEVANCE: Telomeres are the protective DNA-protein caps at chromosome ends that prevent chromosome fusions and degradation of the terminal DNA sequence. Proteins that make up the protective cap structure have to perform different activities as cells go through the cell cycle. During much of the cell cycle, they must hide the chromosome end so that it is not degraded or fused to another chromosome. However, when the chromosome is replicated they have to make the DNA terminus accessible and they play an active role in recruiting factors needed to replicate the telomeric DNA. The proposed research seeks to understand how telomere proteins function individually, and as part of the larger telomere protein complex, to achieve these opposing roles. It also investigates the function of a novel protein that seems to help broken chromosomes by promoting the addition of a new telomere. The research is pertinent to human health because loss of telomere protection leads to chromosome fusions and chromosome rearrangements, the underlying cause of many forms of cancer. Defects in telomerase, the enzyme that maintains telomere length, are also associated with human disease as the resulting telomere shortening can lead to dyskeratosis congenita and pulmonary fibrosis.